体外培养的鸡原始生殖细胞在早期胚胎迁移的研究

发布时间：2018-02-25 00:22

  本文关键词： 原始生殖细胞 转基因 移植 迁移　出处：《广西大学》2016年硕士论文　论文类型：学位论文

【摘要】：原始生殖细胞(Primodial germ cells,PGCs)作为配子的前体细胞,负责将遗传信息传递给下一代。将分离的PGCs经过体外培养扩增以及转基因修饰后,移植到受体胚内能产生生殖系嵌合体,这是生产转基因禽类的一个理想途径。但在实际的研究操作中,由于对PGCs迁移和归巢性腺知识的缺乏,转基因嵌合体的制备方法没有优化,成功效率非常低。本研究旨在探索PGCs在注射到受体胚胎后的迁移机制以及影响其迁移效率的因素,为提高转基因鸡的生产效率提供一定的依据。首先,我们从广西黄羽鸡(yellow feather chicken,YF chicken) 13-15期鸡胚血液中分离PGCs进行体外培养扩增,经转基因修饰后,对获得的能稳定遗传的GFP-PGCs进行鉴定。结果发现,经过体外培养及转基因修饰的PGCs, SSEA-1染色呈阳性,RT-PCR分析结果显示这些PGCs表达了Dazl、Cvh、PouV、Cdh、Nanog基因,说明了我们获得的GFP-PGCs细胞株仍保留其生物学特性。随后,我们将鸡胚随机分成4组,分别孵育50h、55h、60h、65h后移植GFP-PGCs,探索受体胚龄对PGCs迁移的影响。结果发现,受体鸡胚孵育50h移植PGCs的迁移效率较孵育55h、60h、65h组高,孵育时间越长移植PGCs迁移效率越低。在探究移植细胞数对PGCs迁移的影响试验中,鸡胚随机分成四组,孵育50h后分别注射1000、3000、10000、30000个GFP-PGCs,结果显示,移植的最优细胞数为10000个／胚,移植的PGCs少于10000个／胚时,随着移植细胞数的增加,迁移的PGCs增加,当移植的PGCs多于10000个／胚时,迁移的PGCs不再增加。我们将移植10000个PGCs的鸡胚孵出,出壳4d小鸡睾丸内发现外源GFP-PGCs,证明了外源GFP-PGCs能在受体性腺内生长增殖。随后,成熟雄性嵌合体鸡精液DNA PCR结果显示,4个假定嵌合体中有1个呈GFP阳性。最后,我们探究了CXCR4信号通路在PGCs迁移过程中的作用。RT-PCR结果显示,PGCs表达了趋化因子受体Cxcr4基因。此外,我们在移植的细胞滴中分别添加CXCR4信号通路抑制剂AMD3100和WZ811, AMD3100浓度为OnM、10nM、50nM, WZ811为OnM、1nM、2nM,试验结果表明,50nMAMD3100以及1nM、 2nM WZ811能明显抑制PGCs的迁移(P0.05)。证明了SDF-1/CXCR4信号通路参与调控PGCs在早期鸡胚中的迁移和归巢。综上所述,广西黄羽鸡PGCs能在体外长期培养,经转基因修饰后仍保留其生物学特性。受体鸡胚孵育50h为最佳移植时间,每个鸡胚移植10000个PGCs迁移至性腺的PGCs最多,归巢至性腺的PGCs能在受体睾丸中正常增殖生长。SDF-1/CXCR4信号通路参与介导PGCs的体内迁移过程。
[Abstract]:Primodial germ cells (PGCs), the progenitor of gametes, are responsible for transmitting genetic information to the next generation. After in vitro culture, amplification and modification of the isolated PGCs, transplanted into the recipient embryos, the chimerism of the reproductive line can be produced. This is an ideal way to produce transgenic birds, but in practical research operations, due to the lack of knowledge of PGCs migration and homing gonad, the preparation method of transgenic chimera has not been optimized. The purpose of this study was to explore the migration mechanism of PGCs after injection into recipient embryos and the factors affecting its migration efficiency, and to provide a basis for improving the productivity of transgenic chickens. We isolated and amplified PGCs from the blood of chicken embryo of stage 13-15 of Guangxi yellow feather chickenen. After modified by transgenic method, the stable GFP-PGCs was identified. After in vitro culture and transgenic PGCs, SSEA-1 staining positive RT-PCR analysis showed that these PGCs expressed the gene of Dazlus Cvhus PouVu CDHN Nanog, indicating that our obtained GFP-PGCs cell line still retains its biological characteristics. Then, we randomly divided the chicken embryo into 4 groups. The effects of embryo age on PGCs migration were investigated. The results showed that the migration efficiency of PGCs transplanted after 50 h incubation of recipient chicken embryos was higher than that of 55 h incubated chicken embryos at 60 h or 65 h after incubating for 60 h or 65 h, respectively, and the effect of recipient embryo age on PGCs migration was higher than that in recipient chicken embryos incubated for 50 h or 60 h or 65 h. The effect of the number of transplanted cells on PGCs migration was investigated. Chicken embryos were randomly divided into four groups after incubating for 50 hours. After 50 hours of incubation, 30000 GFP-PGCswere injected into 1000 ~ 3000g / g, respectively. The results showed that the optimal number of transplanted cells was 10 000 / embryo. When the transplanted PGCs is less than 10, 000 / embryo, as the number of transplanted cells increases, the migrated PGCs increases, and when the transplanted PGCs is more than 10, 000 / embryo, the migrated PGCs does not increase. We hatch 10, 000 PGCs chicken embryos. Exogenous GFP-PGCswas found in the testis of 4 days out of shell, which proved that exogenous GFP-PGCs could grow and proliferate in the receptor gonads. Subsequently, DNA PCR of mature male chimera chicken semen showed that one of the four hypothetical chimeras was GFP positive. We investigated the role of CXCR4 signaling pathway in PGCs migration. RT-PCR results showed that CXCR4 expressed chemokine receptor Cxcr4 gene. We added CXCR4 signaling pathway inhibitor AMD3100 and WZ811 into the transplanted cells, the concentration of AMD3100 was OnM10 nMU 50nM, and WZ811 was OnM1nMU 2nM. the results showed that P0.05and 50nMAMD3100 and 1nMMAMD3100 significantly inhibited PGCs migration P0.05. it was proved that SDF-1/CXCR4 signaling pathway was involved in the regulation of PGCs in the early stage. Migration and homing in chicken embryos. Guangxi yellow feather chicken PGCs could be cultured for a long time in vitro, and its biological characteristics were retained after transgenic modification. The best transfer time was 50 hours after incubation of recipient chicken embryos, and 10 000 PGCs per chicken embryo transferred to the PGCs of gonad. Homing to gonad PGCs can mediate the migration of PGCs in vivo by normal proliferation and growth. SDF-1 / CXCR4 signaling pathway.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S831

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